The Urticating Setae of Ochrogaster Lunifer, an Australian Processionary

Medical and Veterinary Entomology (2015), doi: 10.1111/mve.12156

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The urticating setae of Ochrogaster lunifer,an Australian processionary caterpillar of veterinary importance

L. E. PERKINS1, M. P.ZALUCKI1, N. R. PERKINS3,4, A. J. CAWDELL-SMITH4, K. H. TODHUNTER5, W.L. BRYDEN4 andB. W.CRIBB1,2 1School of Biological Science, The University of Queensland, Brisbane, Queensland, Australia, 2Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland, Australia, 3AusVet Animal Health Services, Toowoomba, Queensland, Australia, 4Equine Research Unit, School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland, Australia and 5Vetnostics, North Ryde, New South Wales, Australia

Abstract. The bag-shelter moth, Ochrogaster lunifer Herrich-Schaffer (Lepidoptera: Notodontidae), is associated with a condition called equine amnionitis and fetal loss (EAFL) on horse farms in Australia. Setal fragments from O. lunifer larvae have been identified in the placentas of experimentally aborted fetuses and their dams, andin clinical abortions. The gregarious larvae build silken nests in which large numbers cohabit over spring, summer and autumn. The final instars disperse to pupation sites in the ground where they overwinter. Field-collected O. lunifer larvae, their nests and nearby soil were examined using light and electron microscopy to identify setae likely to cause EAFL and to determine where and how many were present. Microtrichia, barbed hairs and true setae were found on the exoskeletons of the larvae. True setae matching the majority of setal fragments described from equine tissue were found on third to eighth instar larvae or exuviae. The number of true setae increased with the age of the larva; eighth instars carried around 2.0–2.5 million true setae. The exuvia of the pre-pupal instar was incorporated into the pupal chamber. The major sources of setae are likely to be nests, dispersing pre-pupal larvae and their exuviae, and pupal chambers. Key words. Lepidoptera, Notodontidae, Thaumetopoeidae, bag-shelter moth, equine amnionitis and fetal loss (EAFL), true setae.

The bag-shelter moth, Ochrogaster lunifer, is found throughout which the larva’s common name ‘processionary caterpillar’ is coastal and inland Australia where its larvae feed on Acacia, derived. The procession breaks into smaller groups and each Eucalyptus and Corymbia spp. trees. Eggs are laid in spring at larva overwinters in or on the ground within a silk cocoon. the base of a trunk or on a twig in the tree canopy. After hatch- Larvae pupate in early spring and adults emerge in mid-spring. ing, large numbers of the gregarious larvae cohabit and produce Records of bag-shelter nests and caterpillars causing adverse a silken nest which may be formed at the base of the tree, on the effects on the health of humans and livestock date back to the trunk or within the canopy (Fig. 1, Figure S1, online). During early 1900s [Froggatt, 1911 (as Teara contraria)]. Ochrogaster summer and autumn, the larvae develop synchronously through lunifer larvae are covered in hairs (Fig. 2A) and cause an irri- a series of up to seven moults and the nest becomes filled with tating dermatitis upon contact with human skin; in addition, living larvae, exuviae and frass. In late autumn, fully grown osteomyelitis, ophthalmia and more severe allergic reactions larvae leave the nest crawling head-to-tail in a long line from have been recorded [Southcott, 1978 (as Ochrogaster contraria

Correspondence: Bronwen W. Cribb, Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: + 61 7 3365 7086; Fax: + 61 7 3364 3993; E-mail: [email protected]

common diagnosed cause of abortion in the thoroughbred breed- ing industry in New South Wales (Todhunter et al., 2009; Car- rick et al., 2014). When pregnant mares ingest O. lunifer larvae or exuviae, setae penetrate the gut and migrate throughout the body, ultimately causing a range of outcomes including focal mucoid placentitis, compromised foals, abortion and peri- natal death (Cawdell-Smith et al., 2012, 2013; Todhunter et al., 2014a, 2014b). Setae and setal fragments of O. lunifer have been described from the gastrointestinal tract, lymph nodes, uterus and liver of experimentally exposed pregnant mares (Todhunter et al., 2014a), and from their aborted fetuses (Todhunter et al., 2014b). Importantly, setae have been identified in the placentas of EAFL clinical cases (Todhunter & Carrick, 2012). The aims of this study were to look for EAFL-associated setae on the larvae and other life stages of O. lunifer, to describe the morphology and measure the density and distribution of such setae, and to evaluate methods to extract setae from environmental samples. Samples of O. lunifer larvae, exuviae, nests, egg masses and soil near to nests were collected from one site at Murray Sunset National Park in Victoria, 14 sites in the Hunter Valley, New South Wales, one site near Monak in New South Wales, and from one site each in the Lockyer Valley and Draper in Queensland, in 2010, 2013 and 2014. Fifty-three samples representing all nesting types were examined by microscopy, and true setae were quantified on 14 samples of larvae and exuviae. Larvae from one nest at the Lockyer Valley site were reared to adults so that pupae and moths could be examined. The instar stage of each larva or exuvia was estimated using the head capsule width and morphometrics published by Floater (1996). Samples were first examined using an Olympus SZX16 stereomicroscope with a DP26 digital camera attachment (Olympus Corp., Tokyo, Japan) and hair types were classified according to Battisti et al. (2011). The long hairs were trimmed off with micro-scissors to reveal the nature of the shorter hairs and intact true setae embedded in the integument in setal fields; this was carried out while the sample was immersed in propylene glycol in order to coat the cut hairs and any dislodged hairs and true setae, and to prevent them from entering the atmosphere where they might have posed a health risk. Areas of interest were then washed in 70% ethanol and prepared as follows for scanning electron microscopy (SEM). Dry samples were vapour-fixed in a closed vial using 2% aqueous osmium tetroxide overnight and then placed under vacuum for at least 48 h, mounted onto aluminium stubs using Araldite® (www.Selleys.com.au) and polymerized for at least 3 h. Samples were then placed under Fig. 1. Schematic of the five nest types built by Ochrogaster lunifer in vacuum until coated to 15–20 nm with iridium. Samples that Australia. (A) Canopy nest. (B) Trunk nest. (C) Tree-hugger nest. (D) contained no or few loose setae were directly mounted onto Hanging nest. (E) Ground nest. Araldite® on aluminium stubs and polymerized, placed under vacuum, and coated as above. Scanning electron microscopy was performed using a JEOL JSM6460 scanning electron micro- Walker); Battisti et al., 2011; van Bockxmeer & Green, 2013]. scope (JEOL Ltd, Tokyo, Japan) at 12 kV in secondary electron Recently, O. lunifer ingestion was shown to cause a condi- imaging (SEI) or backscattered electron (BSE) mode. Individual tion in horses called equine amnionitis and fetal loss (EAFL) hairs and setae were examined using a Zeiss Axioskop com- (Cawdell-Smith et al., 2012, 2013). This condition was first rec- pound microscope (Carl Zeiss AG, Oberkochen, Germany) with ognized in the Hunter Valley (NSW, Australia) in 2004 (Perkins, a Scion FW-1310C camera and VisiCapture software Version 2005; Todhunter et al., 2009) and has since been described 2.0 (Scion Corp., Frederick, MD, U.S.A.). in the Australian eastern states of Queensland, New South The exoskeletons of O. lunifer larvae and exuviae contained Wales and Victoria (Cawdell-Smith et al., 2012). It is the most extensive fields of microtrichia, numerous barbed hairs of

Fig. 2. (A) A final instar larva of Ochrogaster lunifer. (B) Scanning electron micrograph (SEM) of true setae found in setal fields on O. lunifer dorsal abdominal tergites. (C) Two intact setal fields of true setae (SEM). (D) Arrangement of setal fields on an abdominal tergiteof O. lunifer; the setal fields are empty of true setae (SEM). various sizes inserted into sockets, and smaller, loose true setae sample. The centres and edges of setal fields were examined scattered about the surface. As true setae resembled the majority because the density of setae and setal pits sometimes differed of setal fragments seen in equine tissue in cases of EAFL between these areas. A sample of setae was taken from the (e.g. Todhunter et al., 2014a), the present study focused on the Falcon tube of ethanol in which each sample was sonicated and characterization of these. True setae (Fig. 2B) were found on the lengths measured using the stereomicroscope. The lengths larvae and larval exuviae in tight bunches within specialized of 1024 setae from 13 samples were measured. structures of the cuticle (Fig. 2C) described as setal fields or Fully developed eighth instar larvae of O. lunifer had four ‘mirrors’ in other species of Lepidoptera (Battisti et al., 2011). dorsal setal fields (in a 2 × 2 arrangement) and two dorsolateral The true setae ranged in length from 38 μmto828μm(n = 1024 setal fields on each of the nine abdominal segments (Fig. 2D). setae) (Table 1). Pupae and adult moths did not have true Younger instars did not have the full complement of setal fields setae, although female moths carry urticating scales at the end and setae; the number of setal fields and true setae increased of the abdomen. Although pupae do not have true setae, the with the stage of the larva from third to eighth instar. The exuvia from the pre-pupal larva is incorporated into the silken density of setae in the setal fields of whole larvae averaged pupal chamber (Figure S2, online) and true setae were abundant 66 874 setae/mm2 (range: 42 850–93 325 setae/mm2). Data on in that chamber. Egg masses contained urticating scales from the area of setal fields, the density of true setae permm2 and the ovipositing female, as well as barbed filaments (Figure S3, the abundance of setae per larva or exuvia across the different online), but no true setae. nesting types are given in Table 1. A map of the distribution of setal fields was drawn for each Samples were taken from nests and from the ground near nests. of 14 larvae and exuviae representing all nesting types. Each These samples consisted of various mixtures of exuviae, frass, individual sample was then placed in a 20-mL Falcon tube soil and vegetative matter. A quantity of sample was placed in in 70% ethanol and sonicated for several minutes to dislodge a 20-mL plastic centrifuge tube in a biological safety cabinet the true setae from the cuticle. The sample was rinsed in 70% and enough glycerol to cover the sample was added. The sample ethanol and viewed again under the stereomicroscope. The area and glycerol were mixed gently and allowed to stand for a few of the now empty setal fields was measured, and the width of minutes. A drop taken from the top of the glycerol layer was the head capsule was measured to ascertain instar stage. The examined by light microscopy and the presence and size range setal fields from a selected segment were excised and viewed of hairs and true setae recorded. The sample was left to stand under low-magnification SEM. After removal of the true setae for at least 24 h, after which a further drop was taken from the by sonication, pits in which the true setae had been embedded surface and viewed. As the density of glycerol is greater than were visible in the cuticle making up the base of the setal fields. that of true setae, the setae rise to the top and float on the surface The numbers of setal pits (indicating the number of setae) in of the glycerol. squares of 100 × 100 μm were counted and the density per mm2 The exuviae of previous larval instars were retained in the calculated; one to five such squares were examined for each nests of O. lunifer and abundant true setae were found amongst

Table 1. Area of setal fields and density of true setae on larvae and exuviae of Ochrogaster lunifer.

Area of setal Setal Setae, Setal length, Instar Nest type fields, mm2 Ab seg∗ density/mm2 n/sample μm(n)

Larva Third Ground 0.05 9 67 450 3473 NA Larva Seventh Ground 20.95 5 67 700 1 418 100 62–403 (60) Larva Eighth Ground 31.10 7 67 350 2 094 472 43–380 (216) Larva Fourth Tree-hugger 0.21 9 61 875 13 248 65–201 (80) Larva Fifth Tree-hugger 0.82 9 52 950 43 206 59–363 (72) Larva Eighth Tree-hugger 30.72 4 83 050 2 551 523 44–613 (72) Larva Fourth Canopy 0.13 9 42 850 5683 52–206 (88) Larva Seventh Canopy 27.32 1 93 325 2 549 359 55–315 (60) Exuvia Sixth Hanging 1.82 9 34 467 62 609 67–495 (90) Larva Eighth Hanging 36.30 4 66 160 2 401 669 55–389 (73) Exuvia Fourth Trunk 0.35 9 48 189 16 730 61–355 (41) Exuvia Sixth Trunk 2.52 9 31 155 78 641 38–828 (71) Larva Eighth Trunk 31.06 4 80 900 2 512 823 50–358 (100) Larva Third Unknown 0.08 9 52 000 3923 60 (1)

∗Abdominal segment used for measurements. NA, not available. frass, silk and other contents of the nests. In addition, true setae penetration through animal tissue. How small and long setae were found in eight of 12 samples of soil collected near nests. behave after entering the environment warrants further research Microtrichia, hairs and true setae were abundant on the larval as it is likely to affect the level of mare exposure to setae and the O. lunifer exoskeleton from all nest types, but no modified setae subsequent risk for fetal compromise or death. or spines matching published definitions were seen. True setae Setae remaining at the end of an instar stage are retained in the matched the size of the majority of setal fragments seen in the exuviae and new setae are produced at each moult. The invest- equine tissues of EAFL cases, showed a morphology that would ment in setal production increased with the instar of the larva: in facilitate migration through animal tissue, were unattached and the present study, individual third instar larvae were estimated appeared to be easily shed from the cuticles of both whole lar- to have approximately 4000 setae, whereas eighth instar larvae vae and exuviae, and were found in samples of nest material had approximately 2.0–2.5 million setae. Determining the exact and soil from around nests. Therefore, the present investigation larval or exuvial stage using the morphometric regressions pub- focused on these setae as representing the likely agents of EAFL. lished by Floater (1996) was not always possible as the width of However, hairs and microtrichia cannot be ruled out as causes of the head capsule occasionally fell between the widths reported EAFL and further animal studies are needed to test this possibil- for particular instars. Size variability between sexes of the same ity. True setae were found on third to eighth instars of O. lunifer, cohort and between cohorts from different localities has been but not on pupae or adult moths. The setae conform to the clas- found (Floater, 1996), which makes it difficult to compare a sification of true setae by Battisti et al. (2011) and are similar to larva from one nest with a larva from another nest. Nonethe- true setae from northern hemisphere Thaumetopoea spp. (Lepi- less, an increase in setal abundance along with the stage of the doptera: Thaumetopoeidae) caterpillars (Petrucco-Toffolo et al., larva is clear. The average density of 66 874 setae/mm2 in the 2014), which cause various conditions such as dermatitis, con- setal fields of larvae is similar to the 60 000 setae/mm2 reported junctivitis, oropharyngeal inflammation and asthma-like symp- in larvae of the pine processionary moth (T. pityocampa)(Lamy toms in humans and animals (Battisti et al., 2011). The setae are et al., 1982). The present study found that setal density differed inserted into pits in the cuticle and are pointed and detached at between the middle and outer areas of some setal fields; there- the base (proximal end), and thus are easily shed from the integu- fore the pits in these different areas must be counted to provide ment and readily enter the environment. The morphology of true an accurate estimate of setal density. setae was similar across samples from all nest types. The major sources of true setae in the farm environment appear The lengths of setae ranged from approximately 40 to 800 μm, to be nests in which exuviae and living larvae are concentrated, which represents a somewhat broader range than has been the dispersing final instars and the pupal chambers containing reported for northern hemisphere species of processionary cater- the pre-pupal exuviae. After a nest is abandoned by larvae, its pillar (Petrucco-Toffolo et al., 2014). Short setae of < 100 μm structure loses integrity, thereby exposing its contents, including were noticeably distinct from, yet interspersed with, longer millions of setae, to the environment. Some nest types may pose setae within the setal fields as found by Petrucco-Toffolo et al. more of a threat to mares and livestock than others; for example, (2014) for Thaumetopoea pityocampa Denis & Schiffermüller setae and exuviae may disperse further from a nest high in the and Thaumetopoea pinivora Treitschke. The majority of longer tree canopy than from a nest at the base of a trunk, and ground setae measured between 100 and 400 μm, but a few measured nests may retain high densities of setae for longer. The final > 400 μm. The size of setae affects their dispersal throughout the instar larva has by far the most true setae of all the life stages environment (Petrucco-Toffolo et al., 2014) and may affect their (2.0–2.5 million per larva) and a single larva or exuvia may

© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12156 Urticating setae of a processionary caterpillar 5 carry sufficient setae to induce EAFL in pregnant mares. Given van Bockxmeer, J.J. & Green, J. (2013) Paediatric osteomyelitis after the urticarial nature of O. lunifer larvae, they are unlikely to exposure to toxic Ochrogaster lunifer moth. Medical Journal of be knowingly ingested by horses (Cawdell-Smith et al., 2012). Australia, 199, 331–332. Therefore, the entry of setae into the environment from nests Carrick, J.B., Perkins, N.R. & Zalucki, M.P. (2014) Causes of abortion in and the accidental ingestion of exuviae or pupal chambers seem Australia (2005–2012) – proportion of cases due to equine amnionitis the most likely routes of exposure to these animals. Ecological and fetal loss (EAFL). Journal of Equine Veterinary Science, 34, studies are currently underway to determine the dispersal and 212–214. pupation sites of O. lunifer in the horse farm environment and to Cawdell-Smith, A.J., Todhunter, K.H., Anderson, S.T., Perkins, N.R. & determine the means by which horses are exposed. The present Bryden, W.L. (2012) Equine amnionitis and fetal loss: mare abortion following experimental exposure to processionary caterpillars study shows that setae do enter the soil around ground nests and (Ochrogaster lunifer). Equine Veterinary Journal, 44, 282–288. future work will determine the rates of dispersal of setae from Cawdell-Smith, A.J., Todhunter, K.H., Perkins, N.R. & Bryden, W.L. both ground and elevated nests. (2013) Exposure of mares to processionary caterpillars (Ochro- gaster lunifer) in early pregnancy: an additional dimension to equine amnionitis and foetal loss. Equine Veterinary Journal, 45, 755–760. Supporting Information Floater, G.J. (1996) The Brooks–Dyar rule and morphometrics of the processionary caterpillar Ochrogaster lunifer Herrich-Schäffer Additional Supporting Information may be found in the (Lepidoptera: Thaumetopoeidae). Australian Journal of Entomology, online version of this article under the DOI reference: DOI: 35, 271–278. 10.1111/mve.12156 Froggatt, W. (1911) Bag-shelter caterpillars of the family Liparidae that are reputed to kill stock. The Agricultural Gazette of New South Wales, Figure S1. Types of nest built by Ochrogaster lunifer larvae. (A) 22, 443–447. Canopy nest. (B) Trunk nest. (C) Tree-hugger nest. (D) Hanging Lamy, M., Ducombs, G., Pastureaud, M.H. & Vincendeau, P. (1982) nest. (E) Ground nest. Productions tégumentaires de la processionnaire du pin (Thaume- Figure S2. Ochrogaster lunifer pupa and its pupal chamber topoea pityocampa Schiff.) (Lépidoptéres). Appareil urticant et (opened to show the exuvia of the final instar larva inside). appareil de ponte. Bulletin de la Société Zoologique de France, 107, 515–529. Figure S3. (A) Egg mass of Ochrogaster lunifer and second Perkins, N.R. (2005) Equine Amnionitis and Foetal Loss (EAFL) Report. instar larvae. (B) The abdominal tuft scales and barbed filaments Hunter Valley Equine Research Centre, Scone. comprising the egg mass into which the female lays her eggs. Petrucco-Toffolo, E., Zovi, D., Perin, C. et al. (2014) Size and dispersion of urticating setae in three species of processionary moths. Integrative Zoology, 9, 320–327. Acknowledgements Southcott, R.V. (1978) Lepidopterism in the Australian region. Records of the Adelaide Children’s Hospital, 2, 87–173. This research was supported by a University of Queensland Col- Todhunter, K.H. & Carrick, J.B. (2012) Presence of caterpillar setae in laboration and Industry Engagement Fund (CIEF) grant, the the placenta from a field case of EAFL. Proceedings of the Fourth Hunter Valley Equine Research Foundation (HVERF), and an Australasian Equine Science Symposium, 4, 59–60. Australian Research Council (ARC) Linkage Project grant (LP Todhunter, K.H., Perkins, N.R., Wylie, R.M. et al. (2009) Equine 140100687). The authors thank Professor A. Battisti, Depart- amnionitis and fetal loss: the case definition for an unrecognised cause ment of Agronomy, Food, Natural Resources, Animals and of abortion in mares. Australian Veterinary Journal, 87, 35–38. the Environment, University of Padua, Padua, Italy, and col- Todhunter, K.H., Cawdell-Smith, A.J., Bryden, W.L., Perkins, N.R. & Begg, A.P. (2014a) Processionary caterpillar setae and equine fetal leagues for reviewing an earlier version of the manuscript. loss: 1. Histopathology of experimentally exposed pregnant mares. The authors acknowledge the facilities and scientific and tech- Veterinary Pathology, 51, 1117–1130. nical assistance of the Australian Microscopy and Micro- Todhunter, K.H., Cawdell-Smith, A.J., Bryden, W.L., Perkins, N.R. & analysis Research Facility (AMMRF) at the University of Begg, A.P. (2014b) Processionary caterpillar setae and equine fetal Queensland. loss: 2. Histopathology of the fetal-placental unit from experimentally exposed mares. Veterinary Pathology, 51, 1131–1142. References Accepted 1 September 2015 Battisti, A., Holm, G., Fagrell, B. & Larsson, S. (2011) Urticating hairs in arthropods: their nature and medical significance. Annual Review of Entomology, 56, 203–220.